Vibrio cholerae, der Erreger der gastrointestinalen Erkrankung Cholera, ist ein Gram- negatives, fakultativ anaerobes gekrümmtes Stäbchenbakterium und zugleich der wohl bekannteste Vertreter der Familie Vibrionaceae. Es persisitiert die meiste Zeit in aquatischen Ökosystemen wie Flüssen, Seen oder Meeresküsten, wo das Bakterium meist mit Crustaceen oder anderen Organismen mit Chitin-haltigen Oberflächen assoziiert vorliegt. Über orale Aufnahme kontaminierter Lebensmittel oder von Wasser kann das Bakterium in den menschlichen Organismus gelangen und dort den oberen Dünndarmbereich kolonisieren, wo letztlich durch verschiedene Virulenzfaktoren, aber hauptsächlich durch das Cholera-Toxin, die Symptomatik der Cholera ausgelöst wird. V. cholerae ist somit sowohl in seiner natürlichen Umgebung, als auch im humanen Wirt höchst unterschiedlichen Umweltbedingungen ausgesetzt. Diese alternierenden Umweltreize stellen verschiedene Anforderungen an die Expressions- und Regulationsfähigkeiten von Proteinbiosynthesen des Bakteriums dar. Die Notwendigkeit einer raschen Adaption setzt daher vielfältige und komplexe Genregulationsmechanismen voraus. Im ersten Teil der hier vorliegenden Arbeit sollte die Genregulation des chs-Operons untersucht werden. Als Grundlage dienten hierbei Hinweise, nach welchen dieses Operon als putatives PTS eine Rolle für den Metabolismus von dem Chitin-Derivat Chitobiose spielen könnte. Zudem sollte der Einfluss des aus Escherichia coli bekannten Repressors Mlc auf die Expression des Operons tiefer gehend untersucht werden. Im Rahmen dieser Arbeit war es gelungen, das als ChsR benannte Protein eindeutig als spezifischen LacI-ähnlichen Repressor für das chs-Operon zu bestätigen. Weiter konnte auch eine cAMP-abhängige Expressionsinduktion bestätigt werden, welche sich allerdings nur bei inaktiven ChsR durchsetzen kann. Als spezifischer Induktor für den Repressor ChsR konnte Chitobiose (GlcN)2 identifiziert werden, welches zwar bei dem in dieser Arbeit verwendeten O1-Stamm SP27459-S nicht als alleinige Kohlenstoffquelle dienen kann, aber unter induktiven Konzentrationen die Repressoreigenschaft von ChsR inhibiert. Zugleich konnte ChsC als für den Import des Induktors Chitobiose verantwortliches Protein identifiziert werden. Weiter nicht eindeutig zu klären blieb der Einfluss von Mlc auf das chs-Operon. Zwar konnte der aktivierende Effekt von Mlc auf die chs-Expression durch Komplementation bestätigt werden, der genaue Mechanismus bleibt jedoch weiterhin unbekannt und bedarf weiterer Untersuchungen. Einzig der Einfluss von Mlc auf den Chitobiose-Import konnte ausgeschlossen werden. Im zweiten Teil dieser Arbeit sollte der weitaus komplexere Mechanismus der Virulenzgenregulation untersucht werden. Im Fokus stand hierbei der Hauptvirulenz-genregulator ToxR und dessen Abhängigkeit von der periplasmatischen Protease DegS. Anhand unterschiedlicher Experimente auf Promotoraktivitäts-, mRNA- und Proteinebene konnte eine Abnahme der ToxR-Aktivität in der degS-Knockout Mutante beobachtet werden, was auf eine Aktivierung von ToxR durch DegS schließen lässt. Weiter konnte eine Abhängigkeit der Aktivität von ToxR von der ebenfalls DegS-abhängigen RpoE-Signalkaskade ausgeschlossen werden. Auch konnte gezeigt werden, dass die Integrität von ToxR durch ToxS, nicht aber durch DegS bestimmt wird. Der exakte Mechanismus der DegS-induzierten ToxR-Aktivierung konnte im Rahmen dieser Arbeit nicht mehr ermittelt werden. Es wurden jedoch Hinweise darauf gewonnen, dass eine direkte ToxR-DegS-Interaktion im periplasmatischen Raum stattfinden könnte. Die in dieser Arbeit gewonnen Erkenntnisse hinsichtlich der ToxR-Regulation durch DegS bieten sowohl eine interessante neue Perspektive der Funktionsweise der periplasmatischen Protease DegS, als auch eine breite Grundlage für weitergehende Untersuchungen bezüglich der Aktivierung des wichtigsten Virulenzregulators ToxR in V. cholerae. / Vibrio cholerae, the causative agents of the gastrointestinal disease cholera, is a Gram-negative facultative anaerobic curved bacterium. It further is probably the best characterized member of the family Vibrionaceae. V. cholerae mainly persists in aquatic ecosystems such as rivers, lakes or sea-coasts where it is found associated with crustaceae and other organisms exposing chitin-containing surfaces. The bacterium infects the human organism via the oral uptake pathway by ingestion of contaminated food or water. Subsequently, it colonizes the upper part of the small intestine and there it eventually causes the typical symptoms of cholera. Thus, both in its natural surrounding and within the human host, V. cholerae faces dramatically alternating environmental conditions. These challenges exhibit different demands and flexibility to alteration of protein expression. This necessity for efficient adaption requires manifold and complex mechanisms of gene regulation. In the first part of the study presented here, the gene regulation of the chs-operon has been examined. In the forefront of this examination there were indications that this operon may play a role as a putative PTS for the metabolism of the chitin-derivate chitobiose. Furthermore, the influence of the in Escherichia coli well-known repressor Mlc on the expression of the operon has been determined. Within this study the protein termed ChsR could be confirmed as a specific LacI-similar repressor type protein for the chs-operon. Also, a cAMP-dependend induction of expression could be verified, which however, can only be achieved when ChsR is inactive. Chitobiose (GlcN)2 has been identified as the specific inductor for the repressor ChsR. This inductor substrate cannot be used as the only carbon-source for the O1-strain SP27459-S, but is able to act on the repressor ChsR under inductive concentrations to cause depression on the chs-operon. Furthermore, ChsC could be identified to be responsible for the import of the inductor chitobiose. The influence of Mlc on the chs-operon could not be elucidated. Even though the activating effect of Mlc on the chs-expression has been confirmed via complementation analysis, however the exact mechanism remains unknown and needs further investigations. Finally, an influence of Mlc on the import of chitobiose could be ruled out. In the second part of this study a far more complex mechanism of virulence gene expression has been investigated. The examinations concentrated on the main virulence regulator ToxR, which is involved in gene regulation of cholera-toxin genes and others, and its dependence on the periplasmatic protease DegS. On the basis of various experiments a decrease of ToxR-activity in a degS-knockout mutant could be observed on promoter-activity-, mRNA- and protein level, utilizing the ToxR dependent regulated porin OmpU. The obtained results clearly indicated that an activation of ToxR via interaction with DegS seems possible. Furthermore, a dependence of ToxR-activity on the DegS-dependent RpoE-signal cascade could be ruled out. Also it could be demonstrated that the integrity of ToxR is maintained by ToxS, but not by DegS. However, the exact mechanism of the DegS-induced activation of ToxR could not be determined within this study and should be investigated in future. So far only genetic derived indications have been gained that there is direct interaction between ToxR and DegS in the periplasmic space, a proof by protein/protein interaction is still lacking. The findings summarized in this study addressing the regulation of ToxR via DegS present an interesting new perspective of the function of the periplasmic protease DegS involved in affecting a general virulence regulatory pathway. Moreover, the data will serve as the basis for further investigations on the molecular mechanism of activation and signal transduction of the most important virulence factor ToxR in V. cholerae.

The genome of Escherichia coli harbors several catabolic operons involved in the utilization of a wide variety of natural compounds as carbon sources. The chitobiose (chu) operons of E.coli Is involved in the utilization of chitobiose(disaccharide of N-acety1-D-glucosamine) and cellbiose (disaccharide of glucose) derived from the two most abundant naturally occurring carbon sources on earth, chitin and cellulose respectively. The operon consists of the chbBCARFG genes coding for transport, regulation and hydrolysis functions required to utilize these compounds; the chuyBCA genes code for a multi-subuni PTS transporter ; the chuR codes for a dual function repressor/activator of the operon; the chbF codes for a phospho-glucosidase and the chbG codes for a protein of unknown function. The chu operon Is regulated by three transcription factors; NagC, a key regulator of the nag genes involved in amino sugar metabolism; ChbR, a dual function operon-specific regulator; and CRP_cAMP. The operon is repressed by NagC and ChbR in the absence of catabolic substrate. In the presence of chitobiose, expression is induced by the abrogation of NagC-mediated repression by GlcNAc-6-P generated by the hydrolysis of chitobiose-6-P and subsequent activation of transcription by ChbR and CPR-cAMP. Wild type E.coli connot utilize cellbiose due to the inability of cellbiose to induce expression from the operon. The simultaneous presence of a loss of function mutation in nagC and a gain –of-function mutation in chbR is necessary and sufficient to allow cellbiose to induce expression and confer on E.coli the ability to utilize cellbiose. The activation step by ChbR and CPR-cAMP requires an inducer that is recognized by ChbR. The chemical identity of the inducer and the mechanism of transcriptional activation by ChbR and CPR-cAMP are not understood. The studies described in the chapter 2 shows that chbG is essential for the utilization of the acetylated sugars chitobiose and chitotriose while it is dispensable for the sugars lacking the acety1group such as cellobiose and chitosan dimer, a disaccharide of N-glucosamine. ChbG is produced as a cytosolic protein and removes one acety1 group from chitobiose and chitotriose thus shows a mono-decetylase activity. Taken together, the observing suggest that ChbG deacetylates chitobiose-6-P and chitotriose-6-P producing the mono-decetylated from of the sugars. The deacetylateion is necessary for their recognition both as inducers by ChbR to activate transcription along with CRP-cAMP and as substractes by phosop-glucosidase ChbF. Cellobiose positive(Cel+) mutants carrying nagC delection and different gain-of-function mutations in chbR are independent of chbG for induction by chitobiose suggesting that the mutations in ChbR can allow it to recognize the acetylated form of chitobiose-6-P. Despite normal induction, the mutants to grow on chitobiose without chbG are consistant with the requirement of deacetylation for hydrolysis by ChbF. The prediction active site of chbG was validated by demonstrating the loss of chbG function upon alanine substitution of the putative metal binding residues. Vibro cholerace ChbG can complement the function of E.coli ChbG indicating that ChbG is conserved in both the organisms. The studies presented in chapter 3 address the mechanism of transcriptional activation of the chb operon by ChbR and CPR-cAMP. ChbR and CPR-cAMP function in a synergistic manner in response to the induction signal. The synergy is not because of their cooperative binding to the DNA. The role of CRP as a class I activator via the known mechanism involving interaction between the Activation region1 (AR1) and the C-terminal domain of the alpha subunit of RNA polymerase (CTD) was not crucial for the chb operon. A direct interaction between the two activators in virto was observed. Based on these results and the close spacing of the synergy is due to interaction between the two regulators bound to DNA that is enhanced in the presence of the inducer, binding about an optimal confirmation in ChbR required to interact with RNA polymerase. ChbR contacts different residues in the subunit in response to cellbiose and chitobiose; whereas it utilizes the known residues in the presence cellbiose, it appears to require different and unknown residues for induction in the presence of chitobiose. In conclusion, the studies reported in chapter 2 and 3 provide an understanding of the regulation of the chitin oligosaccharides utilization in E.coli at different levels. The broad implications of these studies and possible future directions are discussed in chapter 4. ChbG is an evolutionary conserved protein found in both prokaryotes and enkayotes including humans. ChbG homologs have been implicated in inflammatory bowel disorders in humans and development in metazoans. Therefore, the studies on chbG described in this thesis have been broader significance.

Escherichia coli has the ability to respond to stress such as starvation in a very efficient manner. Under conditions of starvation wherein a novel substrate is provided as a sole nutritional source, Spontaneous mutants arise in a population of E.Coli that are able to utilize this novel carbon Many generic systems, upon mutational activation, have been shown to allow E.coli to Grow on novel substrates. . Wildtype E.coli is not able to utilize cellobiose, a disaccharide of glucose, as a carbon source. However after prolonged incubation with cellobiose as a sole carbon source, spontaneous Cel+ mutants can be isolated. The Cel+ derivatives have mutations in the chb operon involved in the utilization of N-N-diacetylchitobiose, a disaccharide of N-acetyl glucosamine. The chb operon of E.coli is comprised of six ORFs (chbBCARFG) with a ~200bp regulatory region (chbOP); chbBCA encode the IIB, IIC and IIA domains of the PTS-dependent permease respectively, chbR encodes for a dual function activator/repressor, chbF encodes the phopho-chitobiase and chbG codes for a protein of unknown function. It has been shown that the three proteins ChbR, CAP and NagC regulate the expression of the chb operon. ChbR along with CAP activates the chb operon in the presence of chitobiose. In the absence of the inducer, ChbR, along with NagC, represses the chb operon. Activation of the chb operon allowing utilization of cellobiose was earlier shown to occur either via insertion of IS1, IS2 or IS5 into the regulatory region (chbOP) upstream of the transcription start site or by base substitutions in chbR. Comparison of the chb operon sequence obtained from various Cel+ mutants with E.coli K12 genome sequence showed many differences. These differences were clustered in both the permease (chbBCA) as well as the enzyme (chbF) of the chb operon, suggesting that mutations are needed in all the ORFs of this operon in order to alter the specificity of E.coli towards utilization of cellobiose. The main objective of this thesis is to elucidate the mechanism of mutational activation of the chb operon of E.coli to allow utilization of cellobiose. These studies have shown that two classes of mutations, those that abrogate repression by NagC and those that alter the regulation by ChbR, together are necessary and sufficient to confer a Cel+ phenotype to E.coli. These studies also show that the wildtype permease and phospho-â -glucosidase are able to recognize and cleave cellobiose. Initial experiments were designed to study the role of independent mutational events of either insertion within the regulatory region or loss-of-function of chbR in conferring E.coli a Cel+ phenotype. The single mutational event of either the insertion within the regulatory region chbOP that disrupts the strong NagC binding site (mimicking an IS element) or knockout of chbR did not confer on E.c oli a Cel+ phenotype. However the presence of the artificial insertion within chbOP accelerated the process of obtaining Cel+ mutants suggesting a positive role for insertion elements. The apparent inability of the chbR knockout strain to mutate to Cel+ suggested that chbR is essential for acquisition of a Cel+ phenotype. Reporter gene assays showed that the presence of an insertion within chbOP enhances the promoter activity marginally. The role of chbR as a repressor was further ascertained by increased promoter activity seen from wildtype chbOP-lacZ fusion in a chbR knockout strain. A marginal enhancement in promoter activity in the presence of cellobiose in a strain carrying a wildtype chbR as compared to chbR knockout strain suggested an additional positive role of chbR. The inability of cellobiose to produce an inducing signal necessary for activation by wildtype ChbR protein suggested that gain-of-function mutations within chbR locus might play a crucial role in acquisition of cellobiose utilization phenotype by E.coli. The chbR clones obtained from various Cel+ mutants could activate transcription from the chb promoter at a higher level in the presence of cellobiose. However this activation was seen only in a strain carrying disruptions of the chromosomal nagC and chbR loci. These transformants also showed a Cel+ phenotype on the MacConkey cellobiose medium suggesting that the wildtype permease and enzyme upon induction could recognise, transport and cleave cellobiose, respectively. This was confirmed by cloning the wildtype genes encoding the permease and phospho-â -glucosidase under a heterologous promoter (Plac). The wildtype E.coli strain transformed with a plasmid carrying the genes could utilize cellobiose efficiently. Large scale isolation of Cel+ mutants was undertaken. Variation in the ability of cellobiose utilization was observed among the different mutants. Several Cel+ mutants retained the ability to utilize chitobiose. Cel+ mutants lacking insertions within chbOP contained a loss-of-function mutation at the nagC locus. The sequencing of the chbR locus from Cel+ mutant strains showed a single basepair change at the DNA level translating into a single amino acid change when compared to the Cel- counterpart. Nucleotide sequence of chbR obtained from two Cel+ natural isolates of E.coli also showed a single base mutation. The chbR clones from the two mutants, when transformed into a strain carrying disruptions at the chromosomal nagC and chbR loci, conferred it a Cel+ phenotype. Initial characterization of one of the mutant ChbR (N238S) was carried out. Reporter assays in a strain containing a wildtype copy of chbR at the genomic locus and a disruption of nagC showed that the wildtype ChbR is dominant over the mutant ChbR (N238S). The biochemical investigations of the wildtype and mutant ChbR (N238S) were undertaken. Wildtype ChbR showed non-specific binding to chbOP that could not be competed out by excess cold DNA. DNaseI protection assays confirmed that wildtype ChbR formed a relatively nonspecific complex with chbOP as compared to mutant ChbR (N238S). Finally DNaseI footprinting experiments showed that mutant ChbR (N238S) binds the specific direct repeat within chbOP better than the wildtype protein. These results indicated that mutant ChbR (N238S) has lost its ability to repress transcription by its inability to bind chbOP non-specifically. In addition, the mutant ChbR (N238S) has acquired the ability to activate transcription in the presence of cellobiose. This could be partly mediated via enhanced binding of the mutant ChbR (N238S) to the specific DNA binding site within chbOP in contrast to its wildtype counterpart. To conclude, this work has shown that acquisitive evolution of E.coli towards utilization of cellobiose in laboratory conditions alters the regulation of the chb operon and allows it to acquire new metabolic capability for utilizing cellobiose under selective pressure.